Recombinant production of enzymatically active male contraceptive drug target hTSSK2 - Localization of the TSKS domain phosphorylated by TSSK2

Protein Expression and Purification
Jagathpala ShettyJohn C Herr

Abstract

The testis-specific serine/threonine kinase 2 (TSSK2) has been proposed as a candidate male contraceptive target. Development of a selective inhibitor for this kinase first necessitates the production of highly purified, soluble human TSSK2 and its substrate, TSKS, with high yields and retention of biological activity for crystallography and compound screening. Strategies to produce full-length, soluble, biologically active hTSSK2 in baculovirus expression systems were tested and refined. Soluble preparations of TSSK2 were purified by immobilized-metal affinity chromatography (IMAC) followed by gel filtration chromatography. The biological activities of rec.hTSSK2 were verified by in vitro kinase and mobility shift assays using bacterially produced hTSKS (isoform 2), casein, glycogen synthase peptide (GS peptide) and various TSKS peptides as target substrates. Purified recombinant hTSSK2 showed robust kinase activity in the in vitro kinase assay by phosphorylating hTSKS isoform 2 and casein. The ATP Km values were similar for highly and partially purified fractions of hTSSK2 (2.2 and 2.7 μM, respectively). The broad spectrum kinase inhibitor staurosporine was a potent inhibitor of rec.hTSSK2 (IC50 = 20 nM). In vitro phosphoryla...Continue Reading

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Citations

Aug 16, 2016·Fertility and Sterility·Diana L Blithe
Jan 4, 2019·Clinical Chemistry·Jill E LongDiana L Blithe
Jan 23, 2021·The Journal of Clinical Endocrinology and Metabolism·Jill E LongDiana L Blithe

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